"...we are not simply
contending in order that my view or that of yours may prevail, but I presume we
ought both of us to be fighting for the truth..."

From Philebus,
the Dialogues of Plato

SUMMARY

It is currently accepted that
a positive Western blot (WB) HIV antibody test is synonymous with HIV infection
and the attendant risk of developing and dying from AIDS. In this communication
we present a critical evaluation of the presently available data on HIV
isolation and antibody testing. The available evidence indicates that: (a) the
antibody tests are not standardised; (b) the antibody tests are not
reproducible; (c) the WB proteins (bands) which are considered to be coded by
the HIV genome and to be specific to HIV may not be coded by the HIV genome and
may in fact represent normal cellular proteins; (d) even if the proteins are
specific to HIV, because no gold standard has been used and may not even exist
to determine specificity, a positive WB may represent nothing more than
cross‑reactivity with the many non‑HIV antibodies present in AIDS patients and
those at risk, and thus be unrelated to the presence of HIV. We conclude that
the use of the HIV antibody tests as a diagnostic and epidemiological tool for
HIV infection needs to be reappraised.

INTRODUCTION

To date, the only routinely
used methods for demonstrating the presence of HIV in vivo are the ELISA and WB
antibody tests. In the ELISA, the "HIV proteins" are present as a mixture. For
the WB, the HIV proteins are dissociated and placed on a polyacrylamide gel
slab. After electrophoresis, which separates the proteins by molecular weight
and charge, the proteins are transferred to a nitrocellulose membrane by
electroblotting. In performing the antibody test, in both ELISA and WB,the
patient's serum is added to the antigen preparation. It is assumed that if HIV
antibodies are present, they will react with the HIV proteins which, after
washing, are visualised by an enzyme anti‑human‑immunoglobulin chromogen
reaction. In the ELISA the reaction is read optically. For the WB, individual
proteins are recognised and interpreted visually as coloured bands, each of
which is designated with a small "p" (for protein), followed by a number, (which
is the molecular weight in kilodaltons),for example p41 (Fig. 1). The WB is
believed to be highly sensitive and specific, and a positive result is regarded
as synonymous with HIV infection. A positive HIV status has such profound and
far reaching implications that no one should be required to bear this burden
without solid guarantees of the verity of the test and its interpretation. In
this paper, the evolution of the antibody tests, the basis of their specificity,
and the validity of their interpretation are evaluated.

Acceptance of an antibody test
for HIV as being scientifically valid and reliable requires the following:

Once these criteria have been
met, and before the introduction of the antibody tests into clinical medicine,
the test's sensitivity, specificity and predictive values must be determined by
the use of a gold standard, HIV itself.

PROTEINS
CONSIDERED TO BE HIV ANTIGENS

The proteins considered to
represent HIV antigens are obtained from mitogenically stimulated cultures in
which tissues from AIDS patients are co‑cultured with cells derived from
non‑AIDS patients‑‑usually established leukaemic cell lines. Following the
detection of the enzyme reverse transcriptase (RT) in the cultures, the
supernatant, and more often the cell lysates, are spun in density gradients.
Material which bands at 1.16 gm/ml is considered to represent "pure HIV" and
consequently the proteins found at that density are considered to be HIV
antigens.

The immunogenic HIV proteins
are thought to be coded by three genes, namely gag, pol and env. The gag gene
codes a precursor p53/55, which is then cleaved to p24/25 and p17/18. The pol
gene codes for p31/32, and the env gene codes the precursor protein p160 which
is cleaved to p120 and p41/p45.1

The p120 protein. The
generally accepted view is that p120 and p41 are cleavage products of p160,
which is found only in infected cells and not in the virus. However:

(a) p120 is a component only
of the knobs (spikes) on the surface of HIV particles;

(b) The knobs are found only
in the budding (immature) particles; and not in cell free (mature) particles;

(c) immature particles are "
very rarely observed".2

Despite these findings, when
"purified HIV" is tested against AIDS sera, strong bands corresponding to p120
and p160 develop. The solution to these contradictions was found when it was
shown that p80 (vide infra) and "the components visualized in the 120‑160‑kDa
region do not correspond to gp120 or its precursor but rather represent
oligomers of gp41".3

The p41 protein. p41 is one of
the proteins detected by both Gallo's and Montagnier's groups in the first HIV
isolates. However:

(a) Montagnier and his
colleagues observed that AIDS sera reacted with a p41 protein both in HIV and
HTLV‑I infected as well as non‑infected cells, and concluded that the p41 band
"may be due to contamination of the virus by cellular actin which was present in
immunoprecipitates of all the cell extracts"4. Although Gallo's group did not
find such reaction with p41 in non‑infected cells, they did find a p80 protein
and concluded that the reaction was "non‑specific";5

(b) Actin is an ubiquitous
protein which is found in all cells as well as bacteria and several viruses.
Well known retroviruses such as the mouse mammary tumour virus and Rous sarcoma
virus have also been shown to contain actin of cellular origin and it has been
postulated that this protein plays a key role in both retroviral assembly and
budding.6 7 It is also known that oxidation of cellular sulphydryl groups, as is
the case in AIDS patients,8 is correlated with assembly of polymerised actin,9
and that the level of actin antibody binding to cells is determined by the
physiological state of the cells. For this reason actin antibody binding to
cells has been proposed "as a sensitive marker for activated lymphocytes";10

(c) Platelets from healthy
individuals also contain a p41/45 protein which reacts with sera from homosexual
men with AIDS and immune thrombocytopenic purpura (ITP) and which "represents
non‑specific binding of IgG to actin in the platelet preparation".11

The p32 protein. In 1987
Henderson isolated the p30‑32 and p34‑36 of "HIV purified by double banding" in
sucrose density gradients. By comparing the amino‑acid sequences of these
proteins with Class II histocompatability DR proteins,they concluded that "the
DR alpha and beta chains appeared to be identical to the p34‑36 and p30‑32
proteins respectively".12

The p24/25 protein. Detection
of p24 is currently believed to be synonymous with HIV isolation and viraemia.
However:

(a) Apart from a joint
publication with Montagnier where they claim that the HIV p24 is unique, Gallo
and his colleagues have repeatedly stated that the p24s of HTLV‑I and HIV
immunologically cross‑react;13

(b) Genesca et al14 conducted
WB assays in 100 ELISA negative samples of healthy blood donors; 20 were found
to have HIV bands which did not fulfil the then (1989) criteria used by the
blood banks for a positive WB. These were considered as indeterminate WB, (WBI),
with p24 being the predominant band, (70% of cases). Among the recipients of WBI
blood, 36% were WBI 6 months after transfusion, but so were 42% of individuals
who received WB‑negative samples. Both donors and recipients of blood remained
healthy.They concluded that WBI patterns "are exceedingly common in randomly
selected donors and recipients and such patterns do not correlate with the
presence of HIV‑1 or the transmission of HIV‑1", "most such reactions represent
false‑positive results";

(c) Antibodies to p24 have
been detected in 1 out of 150 healthy individuals, 13% of randomly selected
otherwise healthy patients with generalised warts, 24% of patients with
cutaneous T‑cell lymphoma and prodrome and 41% of patients with multiple
sclerosis;15

(d) 97% of sera from
homosexuals with ITP and 94% of sera from homosexuals with lymphandenopathy or
AIDS contain an antibody that reacts with a 25Kd membrane antigen found in
platelets from healthy donors and AIDS patients, as well as a 25 Kd antigen
found in green‑monkey kidney cells, human skin fibroblasts, and herpes simplex
cultured in monkey kidney cells. This reaction was absent in sera obtained from
non‑homosexual patients with ITP or non‑immune thrombocytopenic purpura;11

(e) Conversely, the p24
antigen is not found in all HIV positive or even AIDS patients. In one study,
the polymerase chain reaction (PCR) and p24 were used to detect HIV in patients
at various CDC stages from asymptomatic to AIDS. p24 was detected in 24%
patients and HIV RNA in 50%;16

(f) In another study, "In
half of the cases in which a subject had a positive p24 test, the subject later
had a negative test without taking any medications that would be expected to
affect p24 antigen levels...the test is clinically erratic and should be
interpreted very cautiously".17

The p17/18 protein

(a) In addition to the p24
band, the p17/18 band is the most often detected band in WB of healthy blood
donors;18

(b) Sera from AIDS patients
bind to a p18 protein in mitogenically stimulated HIV infected T‑cells, but not
to non‑infected, unstimulated lymphocytes. However, when the lymphocytes are
mitogenically stimulated, but non‑infected, the AIDS sera bind to a p18 protein
in these non‑infected lymphocytes;19

(c) A monoclonal antibody
(MCA) to HIV p18, reacts with dendritic cells in the lymphatic tissues of a
variety of patients with a number of non‑AIDS related diseases;20 and the "same
pattern of reactivity was present in normal tissue taken from uninfected
individuals as in those taken from HIV positive subjects";21

(d) AIDS patients and those at
risk have high levels of antibodies to the ubiquitous protein‑‑myosin,22 which
has two subunits of molecular weights 18,000 and 25,000.

In view of all the above
evidence it is difficult to defend the view that the bands p41 (and thus p160
and p120), p32, p24 or p18 represent specific HIV proteins. Even if it could be
shown that all these proteins are HIV specific, it cannot be automatically
assumed that antibodies that react with each of these proteins are specific to
HIV infection.

STANDARDISATION OF HIV ANTIBODY TESTS

An antibody test becomes
meaningful only when it is standardised, that is, when a given test result has
the same meaning in all patients, in all laboratories, in all countries. From
the first antigen‑antibody reactions performed by Montagnier's4 and Gallo's23
groups (Fig.2, Fig.3) it was found that: (a) not all of the "HIV proteins" react
with all sera from AIDS patients or even sera from the same patients obtained at
different times; (b) sera from AIDS patients may react with proteins other than
those considered to be HIV antigens. Because of these variable reactions, an
essential requirement was to establish criteria as to what constitutes a
positive WB.

Initially, Montagnier's group
considered p24 sufficient to define a positive WB, whereas Gallo's group
considered p41 sufficient. Most, if not all other laboratories, used the
criteria recommended by the CDC, namely the presence of a band at either p24 or
p41. By 1987 it became obvious that those bands were not HIV specific.
Furthermore, till 1987 "there were as many WB procedures as there were
laboratories doing the assay".24 Since then, all major laboratories have changed
their criteria for WB interpretation but in the United States there are still no
nationally agreed criteria, even among the major laboratories:

(a) In 1987 the Food and Drug
Administration (FDA) licensed a WB kit manufactured by DuPont. The DuPont kit
remains the only licensed WB kit and is used by a minority of laboratories. It
specifies "extremely stringent" criteria for a positive result namely "specific
bands representing three different gene products: p24 (gag), p31 (pol), and an
env band, either gp41, gp120 or gp 160";24

(b) The American Red Cross
defines a positive result as presence of antibodies to at least one gene product
from each of the gag, pol and env genes, without specifying which bands;

(c) The Association of State
and Territorial Public Health Laboratory Directors/Department of Defence/CDC
consider a WB positive if two out of p24, gp41 and gp120/160 are reactive;

(d) The Consortium for
Retrovirus Serology Standardization (CRSS) defines a positive WB as the presence
of antibodies to at least p24 or p31/32, and gp41 or gp120/160.25

All the other major USA
laboratories for HIV testing have their own criteria. For all laboratories, a
negative result requires the absence of any and all bands including bands which
do not represent "HIV proteins". All other patterns which do not satisfy a given
laboratory's criteria for a positive or negative test are regarded as WBI by
that laboratory.

Thus, in the scientific
literature, no strips have been published of a standard positive WB. Fig.1 is
reproduced from the instruction manual of a WB kit manufacturer, Bio‑Rad.
Although given as "Examples of a typical reactive patient serum sample and
reaction with a strong, weak and non‑reactive control" it is also stated, "This
example shows typical reactive patterns only, and is not to be used as a
reference for comparisons with results from unknown serum samples...Patient
samples may show varying degrees of reactivity with different proteins, thus
showing different band development patterns...Each laboratory performing Western
Blot testing should develop its own criteria for band interpretation.
Alternatively, band interpretation may be left to the clinician". In addition to
the obvious problems associated with the lack of standardisation, all of the
above interpretations possess major problems:

When the FDA criteria are used
to interpret the WB, only a minimal number (less than 50%) of AIDS patients have
a positive WB, that is, are infected with HIV. If the criteria of the CRSS are
used, the percentage of AIDS patients testing positive increases to 79%. More
importantly, even when the most stringent criteria are used, 10% of control
samples, which include "specimens from blood donor centers", have a positive
WB.25 As already mentioned, Henderson and his colleagues have shown that p31/32
is a non‑HIV protein. Pinter and his colleagues have shown that p160 and p120
are oligomers of gp41. They have also shown that the WB pattern obtained is
dependent on many factors including temperature and the concentration of sodium
dodecyl sulphate used to disrupt the "pure virus", and concluded: "Confusion
over the identification of these bands has resulted in incorrect conclusions in
experimental studies. Similarly, some clinical specimens may have been
identified erroneously as seropositive, on the assumption that these bands
reflected specific reactivity against two distinct viral components and
fulfilled a criterion for true or probable positivity. The correct
identification of these bands will affect the standards to be established for
Western Blot positivity:it may necessitate the reinterpretation of published
results".26

The finding that the p31/32
band represents a cellular protein, and that p120 and p160 are oligomers of p41,
reduces the criteria of the CRSS and that of the American Red Cross to two
bands, p24 and p41, which according to Colonel Donald Burke are "less than
perfectly specific".27 The above findings reduce the criteria of the Association
of State and Territory Public Health Laboratory Directors/Department of
Defence/CDC to p24 or p41, generally accepted as being non‑specific.

Despite the above evidence,
even at present, the p160, p120 and the p41 bands are considered to represent
distinct viral envelope glycoproteins. In fact, the current WHO guidelines
consider a serum positive for HIV‑1 antibodies if "two envelope glycoprotein
bands (with or without) other viral specific bands are present on the strip".28

To date, AIDS in Africa is
defined on clinical grounds. Recently, the CDC recommended the future inclusion
of serological evidence for HIV infection in the African definition of AIDS. The
test recommended is ELISA,29 which cannot be considered specific. In Russia, in
1990, out of 20,000 positive screening tests "only 112 were confirmed" using the
WB as a gold standard.In 1991, of approximately 30,000 positive screening tests,
only 66 were confirmed.30 In the Latin American and Caribbean AIDS definitions
the "clinical findings of HIV infection" are confirmed "by antibody testing
using ELISA, immunofluoresence or Western blot methods". No criteria are given
for WB interpretation.31

REPRODUCIBILITY

The problems associated with
reproducibility may be best illustrated by two examples. Fig.4 represents WB
strips of a serum specimen from a patient with AIDS, tested by 19 laboratories
that participated in the second CRSS conference on WB test standardisation.25
As can be seen, the band pattern obtained with one and the same serum, varies
from laboratory to laboratory, although all laboratories reported this specimen
as positive. The Transfusion Safety Study (TSS) Group in the USA submitted
approximately 100 patient samples weekly for WB testing to three reference
laboratories over three separate periods of several months. With the 100 patient
samples, they submitted aliquots from four quality control (QC) plasmas, two
positive and two negative. HIV positivity or negativity "was based on the
collective experience with each plasma using: (1) licensed EIA systems of five
manufacturers, (2) an immunofluoresence assay, (3) IB in four reference
laboratories, and (4) a radioimmunopreciptation assay in an additional
laboratory". (EIA=ELISA;IB=WB). The samples were then sent to reference
laboratories which were aware of quality control testing, but "the labels and
codes did not permit identification of the QC specimens as such or linkage to
previous QC specimens". QC1#(+) was submitted 40 times to laboratory A, 5 times
to laboratory B and 45 times to laboratory C.

A special panel of QC samples
was sent to laboratories B, C and an additional laboratory D. The panel
consisted of three aliquots of each of eight samples, including batches
QC#1(+),QC#2(+),QC#3(‑) and QC#4(‑).

Discussing the latter results
the authors state: "Only Laboratory C's reports with the panel were consistent
with the data accrued from all other evaluation of reactivity... Laboratory B
reported the three aliquots of QC#1 (+) as respectively positive on the basis of
three bands (gp41, p55 and p65),indeterminate on the basis of a single band
(gp41), and negative (no bands observed). In addition, all three aliquots of
QC#6(‑)were considered indeterminate because only a single band (gp41) was seen.
Laboratory D reported one aliquot of QC#6(‑) as positive (p15, p24, p32, gp41,
p65) and the other two aliquots as negative (no bands observed). It also
reported a band at p55 for all three aliquots of QC#3(‑)".32

In considering the results
detailed above, one must bear in mind that they occurred in Reference
Laboratories, that is, first class laboratories which constitute only a small
number of the total number of laboratories which perform WB testing in the USA.

In addition, many laboratories
continue to use unlicensed WB kits because of cost and the "stringent criteria
required for interpreting the licensed test".33

SPECIFICITY
OF THE HIV ANTIBODY TESTS

The task of authenticating a
new diagnostic test in clinical medicine requires an alternative independent
method of establishing the presence of the condition for which the test is to be
employed. This method, often referred to as the gold standard, is a crucial sine
qua non, and represents the tenet upon which rests the scientific proof of
validity. The only possible gold standard for the HIV antibody tests is the
Human Immunodeficiency Virus itself. Obviously, the clinical syndrome and the
decrease in T4 cells cannot be considered a gold standard. Although HIV has
never been used as a gold standard there is general consensus that proof of the
specificity of the HIV antibody tests is firmly established. For the ELISA,
Gallo's best figures, obtained from AIDS patients and 297 healthy blood donors,
were 97.7% sensitivity and 92.6% specificity assuming borderline tests as
positive, and using the clinical syndrome as gold standard.34

Colonel Donald Burke and his
colleagues from the Walter Reed Army Institute in the USA are credited as having
most thoroughly researched the problem of defining HIV antibody specificity in a
large population and his data is widely believed to represent the current state
of the art.35 Burke et al36 tested a highly selected healthy subpopulation of
135,187 individuals chosen for a very low prevalence of HIV infection‑‑1/10th
that of a much larger pool of applicants (1.2 million), for US military service.
All applicants were screened with an initial ELISA. All reactive ELISA tests
were repeated in duplicate. Then an initial WB was performed and, if diagnostic
or reactive, a second WB was performed on another fresh blood specimen.
Initially the criteria for a positive and diagnostic WB were the "presence of a
band at 41kd, a combination of the bands 24 and 55kd, or both. Beginning in May
1987, the method of preparing blot strips was modified so that antibodies to
gp120 and gp160 could be detected reproducibly, and criteria for a reactive and
diagnostic blot pattern were changed to those of the Association of State and
Territorial Public Health Laboratory Directors". A positive WB was diagnosed if
and only if the first and second serum samples were diagnostic on WB. All of the
diagnostic WB samples were then assayed with four other antibody tests. A WB was
considered "true positive if all four assays on all available serum samples from
an applicant were reactive and diagnostic", but was considered "false positive
if all four assays on all available serum samples from an applicant were
non‑reactive, non‑diagnostic or both". From the 135,187 applicants, there were
16 positive tests. In one of these, the serum was unavailable for further
testing and one applicant declined to provide a second sample. Serum from 27 of
the 29 samples from the 15 applicants found positive were tested by the four
other antibody tests. Fourteen samples were found positive by all four assays
and all four were negative for one applicant. From this Burke and his associates
calculated the false positive rate as 1 in 135,187 or 0.0007%.

They also speculated on the
implications that this data might hold for their entire population of 1.2
million applicants. They calculated the overall prevalence of 1.48 per 1000 in
the entire pool as equivalent to 200 per 135,187. Assuming that the false
positive rate is the same for the whole population they estimated that since
there will be 200 true positive tests per 135,187 persons of which only one will
be a false positive then the "predictive value of a positive diagnosis in the
program is 99.5%, and a specificity of 99.9%".35 36 Much of Burke's and his
colleagues' reasoning is open to criticism:

1.There is no gold standard
for defining HIV infection. Testing the positive WB in the 15 remaining
applicants against four other antibody tests does not enable an independent
establishment of "true" HIV infection as they are the same test;

2.They define:

(a) the true positive tests as
samples which repeatedly test positive in four similar tests.

(b) the false positive tests
as samples which repeatedly test negative in four similar tests.

The number of samples tested
and the repeats is arbitrarily defined. It would be impossible to say what the
outcome would be if for example the ELISA tests were repeated three instead of
two times or if the samples which tested negative in the first ELISA were tested
again with another ELISA or WB. There are well documented reports in which the
ELISA is negative and the WB positive.37

(c) the false positive rate as
the number of false positive results divided by the number of samples tested.

These definitions bear no
resemblance whatsoever to those described in standard texts.38 The correct
definitions are:‑

(i) A true positive is a
positive test occuring in an individual who is HIV infected as defined by an
independent gold standard;

(ii) A false positive is a
positive test which occurs in an individual who, by application of the gold
standard, does not have HIV infection, (but is not necessarily healthy);

(iii) The false positive rate
in the number of false positive tests as a fraction of total number of
individuals who, by application of the gold standard, are not infected with HIV.

3.The Burke et al premises are
quite the opposite to those of Gallo et al where all positive test results in
healthy individuals are regarded as false positive. Based on Gallo and his
associates' premises we must regard all sixteen cases as false positives as
there is no compelling reason for regarding healthy military applicants as
significantly different from healthy blood donors.

4.Burke's extrapolation to the
entire 1.2 million applicants is invalid. This extrapolation can only be done if
the 135,187 applicants were randomly selected from the entire pool, which they
were not. In the rest of the population the false positive rate may have been
much higher for example as a result of higher concentrations of globulins in
general or of autoantibodies in particular.

Their stated figure of 99.5%
positive predictor value is impossible to arrive at without knowledge of the
sensitivity of the WB test and the prevalence of true HIV infection,38 even if
the specificity and the extrapolation were correct.

5.It is impossible to define
specificity, sensitivity and predictive value with the algorithm used by Burke
and his associates. The best they can do with their algorithm is to determine
the reproducibility of ELISA and WB. In this regard, in Burke's larger study of
1.2 million healthy military applicants, approximately 1% of all initial, 50% of
all repeat ELISAs were positive; and 30‑40% of first WB were positive and 96% of
second WB were positive. In other words Burke's larger study reveals:

(b) 4,000 individuals with two
positive ELISA's followed by a negative WB.

(c) 80 individuals with two
positive ELISA's, an initially positive WB and a negative repeat WB.

This cannot be regarded as a
trivial problem since:

(i) both ELISA and WB are
regarded as highly sensitive and specific.24

(ii) Several thousand healthy
individuals have antibodies that react with "HIV proteins" but who are
ultimately deemed not to be HIV infected;

(iii) Even in the best
laboratories, 80 of Burke's healthy applicants would be diagnosed as HIV
infected since, unlike Burke, only one WB is performed.

The problem becomes even more
serious when one realises that by September 1987 by which time, based on the
antibody tests, a causal relationship between HIV and AIDS was generally
accepted, a single positive ELISA or a positive WB, one band (either p24 or p41)
was sufficient to confirm HIV infection. At present, the general opinion is that
the ELISA tests have a "sensitivity and specificity of over 98%, many
approaching 100%",24 and the CDC AIDS definition "accepts a reactive screening
test for HIV antibody without a confirmation by a supplemental test because a
repeatedly reactive screening test result, in combination with an indicator
disease, is highly indicative of true HIV disease".39 (screening test=ELISA).

Burke et al, like Gallo et al,
determined specificity without reference to sick individuals. The definition of
specificity requires that the test is evaluated in persons who do not have the
disease which is under scrutiny, including sick individuals who have other
diseases where antibodies, some of which may interact with HIV antigens, may be
produced for other reasons. The specificity of the HIV antibody tests must be
determined by testing individuals who are immunosuppressed and/or who have
symptoms and clinical signs similar to AIDS, but who are not considered to have
AIDS or HIV infection. This point is well illustrated by the serological tests
for syphilis. A healthy person who is not infected with Treponema pallidum would
very seldom test positive (false positive). However several authors attest to
the presence in various unrelated disorders of biological false positive tests
to syphilis (BFPS), which may occur in patients with auto‑immune haemolytic
anaemia, systemic lupus erythematosus (SLE), idiopathic thrombocytopenic
purpura, leprosy and in drug addicts. More than 20% of drug addicts test
positive and have the highest incidence of BFPS's.40

Persons with BFPS were also
found "to have a high frequency of other serological abnormalities including
anti‑nuclear factors, autoantibodies, and alterations of gamma globulin". This
led researchers to conclude that "a BFP reaction often is a marker for an
unidentified disorder of the immune system that predisposes to autoimmune
diseases".40 It is of significance that a high proportion (14%) of AIDS
patients were also found to have false positive syphilis serology.41

At least two groups of
researchers raised the possibility that the HIV antibody test in Africans and IV
users may also be a BFP reaction. Jaffe et al42 tested 1129 serum samples from
IV drug users and 89 controls from non‑users. All samples were collected during
1971‑1972 and tested by two commercial ELISAs and WB. Seventeen of the samples
from the IV drug users, but not one of the controls was found positive. They
concluded:

"On the basis of our positive
Western Blot data, it appears that parenteral drug users may have been exposed
to HTLV‑III or a related virus as early as 1971. An alternative but equally
viable explanation is that the HTLV‑III seropositivity detected in these
specimens represents false positive or non‑specific reactions".

Biggar and his colleagues43
found that in healthy Africans the probability of finding a positive HIV
antibody test increased significantly with increasing immune‑complex levels.
They concluded "reactivity in both ELISA and Western Blot analysis may be
non‑specific in Africans....the cause of the non‑specificity needs to be
clarified in order to determine how they might affect the seroepidemiology of
retroviruses in areas other than Africa, such as the Caribbean and Japan".

That a positive WB in all
individuals may represent a BFP reaction is suggested by evidence from both
retrovirology in general and HIV antibody testing in particular.

It is known that all
antibodies including MCA are polyspecific and are capable of reacting with
immunising antigens as well as other self and non‑self components.44 45 In
relation to retroviruses, the scientific literature abounds with data which
convincingly show the widespread presence of non‑specific interaction between
retroviral antigens and unrelated antibodies. Much of this work has appeared as
a result of the search for a viral origin for animal and human neoplasms.46 47
48 49 50

In 1975 Gallo discovered that
patients with leukaemia have widespread infection (antibodies) to a retrovirus
which Gallo claimed to have isolated from cultures and fresh tissues of these
patients and which he named HL23V. Gallo suggested that this virus was
aetiologically associated with the disease but HL23V was later shown to be a
"cocktail" of two monkey viruses. In 1980 Gallo discovered HTLV‑I which he and
his associates claim causes adult T‑cell leukaemia. Up to 25% of AIDS patients
have antibodies to this virus,51 however AIDS patients do not develop leukaemia
any more often than the general population. This can only be interpreted as
either HTLV‑I does not cause adult T‑cell leukaemia or some retroviral
antibodies detected in AIDS patients are non‑specific. In 1986 Essex obtained
serological evidence for, and isolated, another "human retrovirus", HTLV‑IV.
Essex's HTLV‑IV was later shown to be a monkey virus, now called Simian
Immunodeficiency Virus. That a positive WB may not represent proof of HIV
infection but is only a non‑specific marker for AIDS, is suggested by the
following data: In drug addicts there is a strong association between high serum
globulin levels and a positive HIV antibody test and this was the "only variable
which remained significant in a logistic regression model";52 In children, using
WB as a gold standard, hyperglobulinaemia identified HIV infected children with
a specificity of 97%.53 Sixty three sera obtained from 23 patients before and
immediately after immunoglobulin infusion were tested for HIV antibodies using
WB. Of the 63 sera, 52 (83%) were found positive. "Several samples tested in an
HTLV‑III p24 radioimmunoassay were also positive. The amount of antibody
detected was greatest immediately after infusion and decreased between
infusions".54

An individual was given six
5ml injections of donated Rh+ serum, administered at 4 day intervals. "The donor
serum was shown to be negative on HIV antibody and antigen ELISA, so was blood
taken from his wife and child". "Blood taken after the first immunization was
shown to be negative on HIV antibody ELISA and immunoblot assay. After the
second immunization a weak signal on ELISA, slightly above the cut‑off level,
was monitored. After the third immunization the signal was strong and immunoblot
revealed distinct interaction with p17 and p55 proteins. An even stronger signal
was monitored after the fifth immunization. Interaction with p17, p31, gp41, p55
and some other proteins was evident".55

Since: (a) individuals from
the main AIDS risk groups, that is, gay men, drug users and haemophiliacs are
exposed to many foreign substances such as semen, drugs, factor VIII, blood and
blood components; (b) individuals belonging to the above groups commonly develop
infections unrelated to HIV; one would expect these individuals to have high
levels of antibodies directed against antigens other than HIV. In fact at
present, evidence exists that individuals with AIDS, AIDS‑related complex (ARC)
and those at risk, have circulating immune complexes, rheumatoid factor,
anti‑cardiolipin, anti‑nuclear factor, anti‑cellular, anti‑platelet, anti‑red
cell, anti‑actin, anti‑DNA, anti‑tubulin, anti‑thyroglobulin, anti‑albumin,
anti‑myosin, anti‑trinitrophenyl and anti‑thymosin antibodies.22 56
Anti‑lymphocyte auto‑antibodies have been found in 87% of HIV+ patients, and
their levels correlate with clinical status.57 58 Unlike normal sera, 37% of
HIV+ sera were found positive for Type‑D retroviruses, 59 whereas HIV is thought
to be a Lentivirus.

It is also known that (a)
serum IgG levels are higher in Black blood donors than in Caucasians;60 (b) some
risk groups, drug users and gay men are exposed to high levels of mitogenic
agents, semen and nitrites,61 62 and that animals treated with such agents
develop antibodies which react with retroviral antigens.63 That the positive
HIV antibody test may be the result of antigenic stimulation, other than HIV, is
further supported by the following data:

1. HIV is thought to be
transmitted by infected needles, yet a higher percentage of prostitutes who use
oral drugs (84%), than IV (46%), test positive;64

2. "Mice of the autoimmune
strains MRL‑lpr/lpr and MRL‑+/+ made antibodies against gp120". Mice that have
been exposed to T‑lymphocytes from another murine strain were shown to make
antibodies against gp120 and p24 of HIV.65

3.Recipients of negative blood
seroconvert and develop AIDS while the donors remain healthy and seronegative.66

5.While the frequency of
positive HIV antibody tests in healthy blood donors and military applicants is
low, patients with tuberculosis (TB), including those with TB localised to the
lungs, both in the USA70 and Africa,71 have high frequency, up to 50%, of
positive WBs. In the USA72 (26 hospitals studied), patients who are not at risk
of developing AIDS, and who do not have any infectious diseases, have a high
rate of positive WB, (1.3% to 7.8%). The above data may be interpreted either as
proof that HIV is spreading to the heterosexual population or that the HIV
antibody tests are non‑specific. That the latter is the case is suggested by the
fact that by 1988, in the USA,73 only approximately 66 white males were reported
to have had "heterosexually acquired AIDS". By 1992 in New York only 11 men were
reported to have AIDS due to heterosexual infection.74

Rodriguez and his
colleagues75 found that Amazonian Indians who have no contact with individuals
outside their tribes and have no AIDS have a 3.3‑13.3% HIV WB seropositivity
rate depending on the tribe studied. In another study76 they found that 25%‑41%
of Venezuelan malaria patients had a positive WB, but no AIDS. The above data
means either that HIV is not causing AIDS "even in the presence of the severe
immunoregulatory disturbances characteristic of acute malaria", as Rodriguez et
al concluded, or the HIV antibody tests are non‑specific.

The problems associated with
the specificity of the WB could be avoided by use of the only suitable gold
standard, HIV isolation. To date this has not been done and based on the
problems associated with HIV isolation, it may never be feasible.

HIV
ISOLATION

It goes without saying that
virus isolation can be used as a gold standard only if it provides conclusive
genetic, virological and molecular evidence for the existence of a unique virus.
For retroviruses, as a first step towards this goal one must:

(a) find particles with
morphological characteristics similar to other retroviruses; (b) demonstrate
that these particles have a unique set of structural components including RNA
and proteins which belong only to these particles and to no other entity.

Peyton Rous77 is credited
with the discovery and isolation of the first retrovirus. In 1911 he was able to
repeatedly induce tumours in a particular breed of chickens by means of tumour
derived, cell free filtrates. Rous contemplated that either a "minute parasitic
organism" or "a chemical stimulant" might form the basis of his observations;
nevertheless, the tumour inducing filtrates became known as "filterable viruses"
or oncoviruses. In the 1950s, in animal cultures and in fresh tissue, especially
tumour tissue, particles later attributed to retroviruses were readily
detectable with electron‑microscopy (EM). In 1970, the enzyme reverse
transcriptase (RT) which transcribes RNA into DNA, was discovered in
oncoviruses. Because of this, in the 1970's, oncoviruses became known as
retroviruses. In the preceding decade, density gradient centrifugation was
introduced to separate and isolate sub‑cellular particles including viruses.
Because some cellular constituents were found to have the same buoyant density
as viruses, when viruses were isolated from cell cultures, the best results
could be obtained with supernatant fluids which: (a) had high viral
concentration; (b) had low cellular contaminants. This was best satisfied by
non‑cytopathic viruses and by culture conditions which maintained maximum
cellular viability. Most animal retrovirus (exceptions are the so called animal
immunodeficiency viruses) satisfy the above conditions. Taking advantage of the
above retroviral properties, by repeated suspension and sedimentation in sucrose
density gradients, one could obtain, at a density of 1.16 gm/ml, a relatively
pure concentration of retroviral particles‑‑that is, obtain retroviral
particles, separate from everything else, and thus isolate them.78 Nonetheless,
as many eminent retrovirologists point out, contamination of the viral
preparation with virus‑like particles which contain RT, but could be nothing
more than "cellular fragments", microsomes from disrupted cells, "membraneous
vesicles which may enclose other cellular constituents including nucleic acids",
especially when "inadvertent lysis of cells" was induced, could not be
avoided.79 80 81 Because of this, to prove that the material which banded at
1.16 gm/ml contained nothing else but particles with "No apparent differences in
physical appearances", and that the particles were indeed retroviruses, every
retrovirus preparation was further analysed using the following assays:

Unlike animal virus cultures
where the particle concentration is very high, in the AIDS cultures/co‑cultures
the particle concentration is low, so low that both Gallo's and Montagnier's
group had difficulty in detecting them. Unlike most animal retroviruses, HIV is
considered to be a cytopathic virus. If this is so, then cell culture
supernatants will contain many cellular constituents. If, as has been recently
proposed, "a single unique mechanism", HIV induced apoptosis, can account for
T4 cell death,83 then the supernatant must also contain apoptotic bodies, that
is, membrane bound cellular fragments which, (like many retroviruses), bud from
the cell surface. Since the size and composition (some contain pyknotic
chromatin) of the apoptotic bodies vary widely,84 one would expect that some of
these fragments will also band at 1.16 gm/ml. It is significant that the AIDS
cultures/co‑cultures do not have maximum viability, and most if not all claims
of "HIV isolation" have been from cellular lysates. Furthermore and most
importantly, in an extensive search of the AIDS literature no electron
micrographs were found from the material which bands at 1.16 gm/ml; all the
electron micrographs are of particles found in the cell cultures. Thus it is
impossible to be know whether the material‑‑lipids, proteins and nucleic acids,
which bands at 1.16 gm/ml, (the "pure HIV particles"), contains any such
particles whatsoever, and if such particles are present, what is their purity.

The presently available
evidence indicates that only about 20% of the proteins which band at 1.16 gm/ml
are "HIV proteins", the rest are cellular, including beta‑2 microglobulin and
HLA‑DR proteins (4.4%).12 85 Thus, even if particles are present at 1.16 gm/ml
and all the proteins assumed to be HIV are embodied in the HIV particle, the
material which bands at 1.16 gm/ml cannot be considered "pure HIV". Conversely,
"Much of the viral protein secreted from HIV‑infected cells is non‑particulate,
and the proportion of (for example) p24 in virions is a function of the viral
genotype and the age of the culture. In extreme cases, less than one per cent of
the total p24 and gp120 present [in the culture] is in virions".86 In fact, p24
is released from "infected cells independently of infectious virus particles"
and RT.87 88

It must be pointed out that
the terms in the AIDS literature "HIV", "HIV isolation", "pure particles",
"virus particles", "virions" and "infectious particles" have a variety of
meanings and include all of the following, but most often without proof of the
presence of a particle: (a) "RNA wrapped in protein";89 (b) material from the
cell culture supernatants which passes through cell tight filters but through
which organisms such as Mycoplasmas may pass;90 (c) the pellet obtained by
simple ultracentrifugation of the culture supernatant91;( d) recently, very
often, detection in AIDS cultures of p24.92 93

In the first report of "HIV
isolation", Montagnier's group detected in a mitogenically stimulated culture
derived from lymph node biopsies of gay men with lymphadenopathy, "a transient",
"reverse transcriptase activity". In mitogenically stimulated umbilical cord
lymphocytes cultured with supernatant from the above cultures, they reported
type‑C retroviral particles (RVP) in the cultures and RT and antigens which
reacted with pre‑AIDS sera in the material which banded at 1.16 gm/ml.4 Gallo's
group did not consider the detection of the above as representing "true
isolation", "...the virus has not been transmitted to a permanently growing cell
line for true isolation and therefore has been difficult to obtain in
quantity".94 However, although Gallo's group used a permanent cell line for
"HIV isolation", they reported nothing more than the same phenomena as
Montagnier's group. Nevertheless, at present, the detection of the above
phenomena are considered to represent "true isolation" and their finding in a
similar culture is regarded as proof of infectivity.

However, isolation is defined
as separating the virus from everything else and not detection of some phenomena
attributed to the virus (RT, antibody/antigen reactions [WB]); or similar to it,
(particles). Phenomena can only be used for viral detection‑‑even then, if and
only if, the phenomena have been identified as being specific for the virus, by
using the isolated virus as a gold standard. Although this has not been done,
the presently available indirect evidence (that is, evidence that has been
obtained without a gold standard) from both general retrovirology and AIDS
research, indicates that RT, RVP and the antigen/antibody reactions are not
specific for HIV, (or even retroviruses).

The specificity of the
antigen/antibody reactions has already been discussed and will not be further
mentioned. In any case, this reaction cannot be used as a gold standard for the
WB, since a test cannot be its own gold standard.

REVERSE TRANSCRIPTASE

In all HIV research, the
copying of the template‑primer An.dT15 when incubated with the supernatant or
the material which bands at 1.16 gm/ml from the AIDS cultures/co‑cultures is
considered proof of HIV RT activity. In many instances this activity is
considered synonymous with "HIV isolation" and is used to quantify the virus.
However:

1. The same template‑primer is
also copied when incubated with material which bands at 1.16 gm/ml from
leukaemic T‑cell cultures95 and normal non‑infected spermatozoa.96 Both An.dT15
and Cn.dG15 are copied by material which bands at 1.16 gm/ml originating from
normal non‑infected but mitogenically stimulated lymphocytes.95 97

2.An.dT15 is copied not only
by RT but also by two (beta and gamma) of the three cellular DNA polymerases.

In fact, in 1975, an
International Conference on Eukaryotic DNA polymerases defined DNA polymerase
gamma as the cellular enzyme which "copies An.dT15 with high efficiency but does
not copy DNA well".98 Thus, the copying of the template‑primer An.dT15, cannot
be considered synonymous with the presence of HIV RT.

PARTICLE
DETECTION

Retroviruses are enveloped
infectious particles about 100‑120nM in diameter with a core comprising a
protein shell and a ribonucleoprotein complex. Retroviruses are classified into
three Subfamilies‑‑Spumavirinae, Lentivirinae and Oncovirinae. Retroviruses
belonging to the latter Subfamily are divided into Type‑A, B, C and D particles.
Nevertheless, some of the best known retrovirologists do not consider the
finding of "virus‑like particles morphologically and biochemically resembling",
retroviruses, proof of the existence of such viruses.99 In the 1970s, such
particles were frequently observed in human leukaemic tissues,99 cultures of
embryonic tissues,100 101 and "in the majority if not all, human placentas".102
However, they continue to be "an intriguing and important problem that remains
to be solved".103

The particles detected in AIDS
cultures/co‑cultures are considered by all AIDS researchers as being HIV.
However:

1. There is no agreement as to
which Genus or even Subfamily of retroviruses they belong. Sometimes agreement
in not found even within the same group. For example, Montagnier's group
initially reported HIV as a Type‑C oncovirus,4 then a Type‑D oncovirus104 and
subsequently as belonging to a different Subfamily of
retroviruses‑‑Lentivirinae.105 Moreover, the "HIV particles" in monocytes
differ from both the Type‑C Oncoviruses and Lentiviruses.106

2. Despite the above,
Gelderblom et al put forward an HIV model (Fig. 5) which has a well defined
morphology and composition, including surface knobs made of p120, a protein
considered to play a crucial role in cytopathogenesis and to be indispensable
for HIV infectivity.107 The model has been accepted and is well known. However,
the same group using EM and immune electron‑microscopy has shown that: (a) knobs
are found only in immature (budding) particles. Immature particles are "very
rarely observed", and are seen only "on metabolically impaired cells";2 108 (b)
mature particles are "hardly, if at all, labelled" by AIDS and ARC sera.
Immature particles are "highly labelled", but so is the rest of the cell from
which they are budding, which "might be due to the fact that natural immune sera
are indeed polyspecific";2 109 (c) like sera, antibodies to p120 react
preferentially with immature particles.107 MCA against gag proteins label the
mature particles, but they also label HIV‑2 particles and simian
immunodeficiency virus particles;110 (d) in the HIV particles, including its
membrane, they111 as well as others,112 detected many cellular proteins, but
with the possible exception of the "lateral bodies", these proteins are not
included in the idealised HIV model.

3. The T‑cell and monocyte
"HIV infected cultures" contain in addition to particles with the morphologies
attributed to HIV, many other "viral particles" unlike any of the "HIV
particles".106 111 113 114 Non‑HIV infected H9 cells, from which most of the
published EM have originated as well as other cells used for "HIV isolation",
CEM, C8166, EBV transformed B‑cells, and cord blood lymphocytes, express budding
virus‑like particles albeit they are somewhat different from particles accepted
as HIV.115 The above data raises questions not only in regard to the origin and
role of the "non‑HIV particles", but also the "HIV particles", and as to which,
if any of these particles, band at 1.16 gm/ml.

4. Budding and mature type‑C
particles appear in metabolically impaired but non‑HIV infected lymphoma
cells.116 "Retroviral particles" antigenically related to HIV have been found
in cultures of salivary gland extracts from patients with Sjorgen's syndrome117.

The independent finding of
"virus‑like" particles in the lymph nodes of AIDS patients with
lymphadenopathy118 and of proteins in the lymph nodes which reacted with MCA to
p55, p24 and p18119 were interpreted as proof that the " virus‑like particles"
were HIV. However:

(a) MCA to p18 react with
lymphatic tissues of patients who suffer from a number of non‑AIDS related
diseases, and also healthy individuals;20 21

(b) As in the AIDS
cultures/co‑cultures, in the lymph nodes of patients with AIDS and persistent
generalised lymphadenopathy, in addition to the "HIV particles", particles
unlike those of HIV are also found;120

(c) Most importantly, in the
only EM study121, either in vivo or in vitro, in which suitable controls were
used and in which extensive blind examination of controls and test material was
performed, virus particles indistinguishable from HIV were found in a variety of
non‑HIV associated reactive lymphadenopathies leading the authors to
conclude:"The presence of such particles do not, by themselves indicate
infection with HIV".

COMMENTS ON
"ISOLATION"

One can conclude then that
neither the antigen/antibody reaction, nor the particles nor RT can be
considered specific for retroviruses. Even if they were, their finding cannot be
considered as synonymous with the detection of an externally acquired
retrovirus, as is claimed to be the case for HIV. Such findings may represent
the expression of endogenous retrovirus (vide infra) or other exogenous
retrovirus.

Lately, "several laboratories
reported retroviral activity [RT, particles] in cells of patients who appear not
to be infected by HIV", an activity said to be "from endogenous retrovirus".122

The cell line most often used
in AIDS research is the leukaemic cell line H9. It is known that H9 is a clone
of HUT78, which was derived from a patient with adult T‑cell leukaemia. Since
the causative agent of this leukaemia is accepted to be HTLV‑I, another
exogenous retrovirus, the H9 cultures should have both RT and retroviral
particles even in the absence of HIV. Because about 25% of AIDS patients have
antibodies to HTLV‑I, about 25% of cultures should have in addition to particles
and RT, a positive WB to HTLV‑I. However, since the proteins from HIV and HTLV‑I
share the same molecular weights, the HTLV‑I WB bands will appear to be positive
for HIV.

A more direct problem
associated with the use of "HIV isolation" as a gold standard is the fact that,
irrespective of the various phenomena accepted by AIDS researchers as
representing "HIV isolation", and despite the fact that no effort has been
spared, it is not possible to "isolate HIV" from all antibody positive patients.
The success rate varies between 17% and 80%.92 93 123 Conversely, when the same
effort is made, HIV can be isolated from some non‑AIDS seronegative patients,
and even from normal seronegative individuals at no risk for HIV infection.124
125 With a more recent method used for "HIV isolation", detection of p24 in
cultures with whole unfractionated blood,126 127 positive results have been
reported in 49/60 (82%) of "presumably uninfected, but serologically
indeterminate" individuals and in 5/5 "seronegative blood donors".128

As far back as 1988,
researchers at the CDC in the USA realised that no correlation exists between
"HIV isolation" and a positive antibody test (which they call documented
infection), and more importantly, between "HIV isolation" in vitro and its
presence in vivo‑‑"correlation between these two methods is limited; they are
inconsistent, in that virus cannot be detected in every person with a documented
infection. Furthermore, the culture technique determines the ability of infected
cells to produce virus in vitro but does not necessarily indicate the status of
virus expression in vivo".129

GENOMIC
INVESTIGATIONS

In the decades following Rous'
experiments, Rous as well as other researchers performed similar investigations
with several animal species. However, although neoplasia could be induced by
injection of filtrates from tumour tissues, (infectious retroviruses, exogenous
retroviruses), no epidemiological evidence existed to suggest an infectious
origin of cancer. In 1939 Andrews "speculated on the possible activation of
latent viral infectious particles in cancerous tissues", and in 1948 Darlington
postulated "that such viruses [endogenous viruses] could arise from cellular
genetic elements which he named proviruses".80 In the 1950s and 1960s the
following experimental evidence was considered proof of the proviral
hypothesis:(a) healthy animals in which no complete virus could be detected had
viral antigens similar to those of exogenous virus; (b) DNA genomes or partial
genomes of the infectious retroviruses were found to be integrated into the
genomes of normal non‑virus producing cells; (c) "Final proof came with the
isolation of infectious viruses from uninfected cells". Healthy non‑virus
producing cells when cultured were found to spontaneously produce viruses.80
Their appearance and yield could be increased a millionfold by (i) mitogenic
stimulation;130 (ii) co‑cultivation techniques;131 (iii) cultivation of cells
with supernatant from non‑viral producing cultures.132 (Note:For HIV isolation,
mitogenic stimulation is an absolute requirement, and in fact, in most cases,
all of the above are employed).

At present it is generally
accepted that "one of the most striking features that distinguishes retroviruses
from all other animal viruses is the presence, in the chromosomes of normal
uninfected cells, of genomes closely related to, or identical with, those of
infectious viruses"80.

Depending on conditions, the
provirus genome remains unexpressed or part or all of it may be expressed. The
latter may or may not lead to the assembly of viral particles (endogenous
retrovirus). 80 In other words, the finding of a viral genome (DNA) or even of
RNA, antigens and antibodies to them, is not proof of the presence of infectious
particles. Although most of the above findings are from animal experiments, at
present, evidence exists that "The human genome carries DNA sequences related to
endogenous retroviral genomes that are subdivided into families according to
sequence homology. Some are present in only a few copies, whereas others are
present in hundreds to thousands of copies".133 Animal data also shows that new
retroviruses may arise by (a) phenotypic mixing; (b) genetic recombination and
deletion. When a cell contains two proviruses, progeny may be found that possess
the genome of one but the structural proteins of either or both viruses present.
Conversely, the RNA may be viral but at least some of the proteins may be
cellular. In other instances, the particles do not have a genome at all, or one
or more genes are missing (genetically defective viruses). The genetic mixing
can be between viral genomes or between viral and cellular genes.80 134
According to distinguished retrovirologists such as Weiss and Temin, new
retroviral genomes may arise by rearrangement of cellular DNA caused by many
factors including pathogenic processes, a view that proposes retroviruses as an
effect and not the cause of disease.135 136 The time and appearance of the
viral genome "may be millions of years in germ‑line cells and days in somatic
cells".136 In addition to the above, the retroviral replicative cycle "involves
three distinct steps:reverse transcription, DNA polymerization, and the
synthesis of RNA from a DNA template (transcription). Any errors made by the
polymerase enzyme during the first and the third steps are not subjected to
proof reading, the result being pronounced sequence variability".137 Hence, as
long ago as 1973, it was concluded that the above phenomena "will prove a
stumbling block to any genetic analysis of RNA tumour viruses"138 (RNA tumour
viruses=retrovirus). To date, the data on the HIV genome has not altered the
above prediction and shows that many problems may exist with the use of the
genomic studies in efforts to prove infection of AIDS patients with a unique
exogenous retrovirus, HIV. Some of these problems can be summarised as follows:

1.No two HIV genomes are the
same. (a) No two identical HIV have been isolated even from the same person. In
one case where two sequential isolates were made 16 months apart, none of the
provirus in the first isolate was found in the second139 leading one HIV
researcher to conclude "The data imply that there is no such thing as an [AIDS
virus] isolate"140; (b) from the same person at a given time more than one HIV
can be isolated141 142; (c) many, if not all of the proviruses detected in vivo
and in vitro are defective;143 (d) In one and the same patient, the genomic data
in monocytes differs from that in T‑lymphocytes;144 (e) the genetic data
obtained in vitro does not correlate with the data obtained in vivo‑‑"To culture
is to disturb"145; (f) The type of virus isolated is determined by the cell
types used for HIV isolation.142 146

2. There is no correlation
between "isolation" of HIV and detection of the HIV genome. Cultures positive
for "infectious virus", may be "polymerase chain reaction‑‑negative".147

3. HIV sequences cannot be
found in all AIDS patients.

Gallo and his colleagues,
summarising the first hybridisation studies with fresh tissue concluded: "We
have previously been able to isolate HTLV‑III from peripheral blood or lymph
node tissue from most patients with AIDS or ARC" [approximately 50% of patients
referred to by Gallo]. "However, as shown herein, HTLV‑III DNA is usually not
detected by standard Southern Blotting hybridization of these same tissues and,
when it is, the bands are often faint...the lymph node enlargement commonly
found in ARC and AIDS patients cannot be due directly to the proliferation of
HTLV‑III‑infected cells...the absence of detectable HTLV‑III sequences in
Kaposi's sarcoma tissue of AIDS patients suggests that this tumor is not
directly induced by infection of each tumor cell with HTLV‑III...the observation
that HTLV‑III sequences are found rarely, if at all, in peripheral blood
mononuclear cells, bone marrow, and spleen provides the first direct evidence
that these tissues are not heavily or widely infected with HTLV‑III in either
AIDS or ARC".148 These studies were confirmed by many other researchers.

To improve detection, the
polymerase chain reaction (PCR) method was introduced. However, "a striking
feature of the results obtained so far" with this method, as with the standard
hybridisation technique, "is the scarcity or apparent absence of viral DNA in a
proportion of patients"149 and, when viral RNA or DNA is found, the "signal" is
very low. For example, HIV is thought to be transmitted primarily by sexual
intercourse yet with the PCR the "HIV genome" can be detected in a minority of
semen samples (1/25).147 It must be pointed out that a positive PCR, even if
found in all patients as is claimed in some publications,149 cannot be regarded
as signifying the presence of the whole HIV genome. With the PCR "only small
regions may be amplified, a gene at best" 143 that is, one does not detect the
whole viral genome, and, since most HIV "isolates" to date are defective,
detection of part of or a whole gene, or even several genes, cannot be
considered synonymous with the whole HIV genome. Furthermore, the PCR is not
standardised and to date, there has been only one study in which the
reproducibility, sensitivity and specificity of PCR were examined. In this
study, the gold standard used was serological status. Specificity was determined
by measuring the percentage of negative PCR results in seronegative (ELISA),
healthy, low risk individuals (blood donors). The PCR was found not to be
reproducible and "false‑positive and false negatives results were observed in
all laboratories (concordance with serology ranged from 40% to 100%). In
addition, the number of positive PCR results did not differ significantly
between high‑ and low‑risk seronegatives".150

4. The positive hybridisation
results may not be HIV specific.

In 1984 when Gallo and his
associates conducted their first hybridisation studies, they found that when the
results were positive, the hybridisation bands were "faint", "low signal". The
"low signal" was interpreted as proof that HIV infected individuals contain
provirus in small numbers of peripheral blood mononuclear cells and at low copy
numbers. However, according to Gallo and his associates, "theoretically this low
signal intensity could also be explained by presence of a virus distantly
homologous to HTLV‑III in these cells".148

Data which has come to light
since then suggest this theoretical possibility may be a fact:

(i) Although it is no longer
accepted that HIV is transmitted by insects, in 1986 researchers from the
Pasteur Institute found HIV DNA sequences in tsetse flies, black beetles and ant
lions in Zaire and the Central African Republic.151

(ii) In 1984 Gallo's group
reported that the genome of HIV hybridises with the "structural genes (gag, pol,
and env) of both HTLV‑I and HTLV‑II".152 Presently available evidence shows
that normal human DNA contains retroviral genomic sequences related to HTLV‑I
and II.153 154

(iii) In 1985 Weiss and his
colleagues reported the isolation, from the mitogenically stimulated T‑cell
cultures of two patients with common variable hypogammaglobulinaemia, a
retrovirus which "was clearly related to HTLV‑III/LAV"; evidence included
positive WB with AIDS sera and hybridisation with HIV probes.155

(v) Horowitz et al, "describe
the first report of the presence of nucleotide sequences related to HIV‑1 in
human, chimpanzee and Rhesus monkey DNAs from normal uninfected individuals".
They have "demonstrated the presence of a complex family of HIV‑1 related
sequences" in the above species, and concluded that "Further analysis of members
of this family will help determine whether such endogenous sequences contributed
to the evolution of HIV‑1 via recombination events or whether these elements
either directly or through protein products, influence HIV pathogenesis".157

That the positive
hybridisation signals may be due to such events induced by the oxidative agents
(mutagens and mitogens) to which the AIDS risk groups and the cultures are
exposed is suggested by the following: (a) a positive PCR reverts to negative
when exposure to risk factors is discontinued;158 159 (b) monocytes from HIV+
patients in which no HIV DNA can be detected, even by PCR, become positive for
HIV RNA after cocultivation with normal ConA‑activated T‑cells".160

As far back as 1989
researchers at the Pasteur Institute concluded that "the task of defining HIV
infection in molecular terms will be difficult".145 They confirmed their
conclusion in a recent study where they "described the enormous heterogeneity
found in vivo within HIV‑1 populations" and the possibility "that an HIV carrier
may harbour easily in excess of 1010 proviruses, most of which will be
genetically unique". They conclude: "It is therefore possible that the sheer
size of variants within an infected individual will allow HIV to explore totally
new genetic possibilities". The appearance of "radically different genetic"
retroviral structures may be the result of "rearrangement, duplication, deletion
or hypermutation. The transduction of host cell DNA represents possibly the most
startling genetic trait of retroviruses".161

CONCLUSION

It is axiomatic that the use
of antibody tests must be verified against a gold standard. The presently
available data fail to provide such a gold standard for the HIV antibody tests.
The inescapable conclusion from the above discussion is that the use of HIV
antibody tests as predictive, diagnostic and epidemiological tools for HIV
infection needs to be carefully reappraised.

50. Barbacid, M., Bolognesi,
D. and Aaronson, S.A. 1980..Humans have antibodies capable of recognizing
oncoviral glycoproteins:Demonstration that these antibodies are formed in
response to cellular modification of glycoproteins rather than as consequence of
exposure to virus. Proc. Natl. Acad. Sci. 77:1617‑1621.

2A: "Cord blood T‑lymphocytes
infected with virus" (HIV‑1) were lysed and the supernatant of a 10,000g
centrifugation of the cell lysate was immunoprecpitated with sera from patients
with lymphadenopathy (P); a healthy donor (h); goat antiserum to HTLV‑I p24 (G);
normal goat serum (g).

2B: As 2A but cells infected
with HTLV‑I instead of HIV‑1.

2C: The cell free supernatant
from the cultures of "cord blood T‑lymphocytes infected with virus" (HIV‑1) was
ultracentrifuged for one hour at 50,000 rev/min.The pellett was banded in
sucrose density gradients. Material which banded at 1.16gm/ml (the complete
virus) was immunoprecipitated with the above sera but instead of normal goat
serum, serum from another healthy donor (h) was used. Although in the published
strips it is hard if not impossible to distinguish any bands, in the text, it is
stated that "three major proteins could be seen: the p25 protein and proteins
with molecular weights of 80,000 and 45,000" (Modifed from Barré‑Sinoussi et al.
Science Vol 220:p870).

Figure 3.

3A: "Lysates of HTLV‑III
producer" H4 clone cells, derived from the HUT78 cell line immunoprecipitated
with various sera.

3B: "Lysates of HTLV‑III
producer" H17 clone cells also derived from the HUT78 cell line,
immunoprecipitated with various sera; (the serum in 3B lane 2 is identical to 3A
Lane 4).

3C: Lysates of H17 and H4
clones (b) "before" and (a) "after infection", immunoprecipitated with serum
from a male heterosexual drug user with lymphandenopathy and thrombocytopenia
(pre‑AIDS). This is the same serum as 3B Lane 5.

3D: "Lysates of H4/HTLV‑III...
cells" (C), or "virus purified from the cells culture fluids", (V), using
(I)‑same serum as 3B Lane 5; (II)‑serum from a patient with pre‑AIDS; (III)
serum from a patient with AIDS. This is the same serum as 3B Lane 4.